Polypeptide purification method

ABSTRACT

This invention relates to a purification method, particularly for purifying tyrosine kinase receptor related polypeptides, and to products made by the method. The method comprises a method of producing tyrosine kinase receptor-related polypeptides, the method comprising expressing a tyrosine kinase receptor-related polypeptide in a recombinant expression system and separating expressed monomeric tyrosine kinase receptor-related polypeptide from multimeric form(s) of the expressed polypeptide in a separation step, the separation step allowing refolding of the expressed tyrosine kinase receptor-related polypeptide into a biologically active form.

FIELD TO WHICH THE INVENTION RELATES

This invention relates to a method for purifying polypeptides and toproducts obtained by the method. In particular, though not exclusively,the invention relates to a method for purifying polypeptides which haveto fold before they are biologically active such as the tyrosine kinasereceptors TrkA, TrkB and TrkC and biologically active variants andportions thereof (all referred to as “tyrosine kinase receptor-relatedpolypeptides”).

BACKGROUND

The tyrosine kinase receptors TrkA, TrkB and TrkC bind neurotrophins.TrkA is biologically active in that it binds nerve growth factor (NGF)with high affinity. It is also biologically active in that it bindsneurotrophin-3 (NT3) with high affinity. TrkB and TrkC bind otherneurotrophins. TrkB binds brain derived neurotrophic factor (BDNF) andneurotrophin-4 (NT4) with high affinity. TrkC binds NT3 with highaffinity. The identification, cloning and sequencing of TrkB and TrkCare described in U.S. Pat. No. 6,027,927. Each receptor moleculecomprises a number of regions or domains. The immunoglobulin-likedomains (Ig) of the tyrosine kinase receptor molecule are of particularinterest in therapeutic applications. More particularly, as disclosed inthe applicant's co-pending patent application, WO99/53055, TrkAIg₂ andvariants, such as the splice variant TrkAIg_(2.6), have therapeuticapplication.

There is a need to produce polypeptides derived from the tyrosine kinasereceptors on a large scale, particularly for therapeutic applications.Production of recombinant polypeptides in bacterial expression systemsis advantageous for several reasons, particularly because relativelyhigh yields of polypeptide can be obtained. Typically yields can be tentimes higher than in human cell systems.

However, the expressed polypeptides such as TrkAIg₂, the secondimmunoglobulin-like domain of TrkA, are difficult to work with in thatthey tend to be produced as a mixture of monomer, dimer and aggregate(i.e. aggregated dimer which may include monomer amongst the dimer) Inparticular in the case of TrkAIg₂, but also in the cases of TrkBIg₂ andTrkCIg₂, only the monomer is, however, active and thereforetherapeutically useful. As discussed in Robertson A. G. S. et al(Biochemical and Biophysical Research Communications 282 (1): 131-141Mar. 23, 2001) dimers of TrkAIg₂ are not able to bind NGF and are notbiologically active. It is likely that small amounts of dimer oraggregate seed the production of more aggregate leading to a decrease inamount of biologically active monomer. This is unusual, many proteinsexist in an equilibrium between monomer, dimer and even tetramer, (e.g.human growth hormone). The removal of dimer is crucial to a long termstable preparation, which is a requirement for pharmaceuticalformulation. Like many proteins, correct conformation of the tyrosinereceptor-related polypeptide is important for biological activity. Whenexpressed in a bacterial inclusion body the polypeptide is folded, butin an incorrect biologically inactive conformation. After expression ina recombinant system the polypeptide must be folded again to achievethat correct conformation. This further folding after expression issometimes referred to as “refolding”.

We are only aware of two other groups that have tried to makerecombinant TrkAIg₂ in bacterial cells. Ultsch et al (J Mol Biol (1999)290, 149-159) made TrkAIg₂, TrkBIg₂ and TrkCIg₂. They made the TrkAIg₂as soluble protein, rather than in inclusion bodies, purified it by ionexchange, then hydrophobic interaction, ion exchange again and gelfiltration. The gel filtration step here was not to allow refolding ofthe polypeptide—it would be assumed that the polypeptide was alreadycorrectly folded as it was in soluble form. TrkBIg₂ and TrkCIg₂,although expressed in the same way, were insoluble. They weresolubilised in urea and dialysed to allow refolding and further purifiedby ion exchange. Solution of crystal structures of the resultingTrkAIg₂, TrkBIg₂ and TrkCIg₂ revealed strand swapped dimers i.e. dimerswhere strand A of one monomer is paired with strand B of another monomer(see the accompanying FIG. 1). FIG. 1 shows a strand swapped dimerconsisting of two TrkAIg₂ monomers in which the A strand from monomer Xunfolds and binds with the B strand from monomer Y. Conversely the Astrand from monomer Y unfolds and binds with the B strand from monomerX. These are inactive. In their discussion it was indicated that none ofthe dimers produced were capable of binding to the natural ligands, incontrast to the domains expressed as immunoadhesins in 293 cells (Urferet al (1995) EMBO Journal 14, 12, p2795-2805). The apparent NGF bindingactivity of the TrkAIg₂—immunoadhesin molecule constructs in animalcells was probably due to the large immunoadhesion scaffold (the F_(c)portion of IgG) holding the TrkAIg₂ region in a correct conformation,which would lead to extensive glycosylation, and would also be likely tosignificantly affect the binding of the construct to other molecules.

Windisch et al (Windisch, J M. et al (1995) J. Biol. Chem. 270 47p28133-28138) produced TrkA derivatives including TrkAIg₂ as maltosebinding protein fusion constructs which were inactive and thereforewould not be suitable for therapeutic use. Maltose binding proteinconstructs are used with “difficult” proteins. It was assumed that theconstructs had folded correctly but it is now apparent that this was notthe case.

Wiesmann et al (Nature, 9 Sep. 1999 401, 184-188) could only produce aco-crystal of NGF and TrkAIg₂ by adding together NGF and TrkAIg_(1,2)i.e. a polypeptide comprising both Ig-like domains of TrkA. Over aperiod of many months the TrkAIg₁ region was ‘nibbled’ away leaving onlythe TrkAIg₂ region bound to the NGF. Such a method is not suitable forcommercial level production of tyrosine kinase receptor relatedpolypeptides.

One method of producing biologically active portions and derivatives oftyrosine kinase receptor-related polypeptides in inclusion bodies inEscherichia coli is disclosed in Holden et al (Holden, P. H. et alNature Biotechnology, 15, July 1997 page 668-672). This method involvesa dialysis step, after extraction of the polypeptide from the inclusionbodies, to allow the expressed polypeptide to refold, and results in ayield of only about 16 mg/litre of polypeptide. WO99/53055 discloses asimilar method of purifying portions and derivatives of TrkA, includingTrkAIg₂, from E. coli inclusion bodies in which the extractedpolypeptide is also dialysed. This method leads to a yield of about ˜50mg/litre. This method produces a product which is relatively unstable,having to be snap frozen after production, and before it can be usedfurther.

As noted above, the methods described in Ultsch et al, WO99/53055 and inHolden et al involve a dialysis step. Dialysis is relativelydisadvantageous in that it requires large amounts of dialysis buffer inorder to limit the concentration of polypeptide (usually to about 0.1mg/ml) and to limit aggregation of the polypeptide. The amount ofdialysis buffer involved precludes the use of a method of producingtyrosine kinase receptor-related polypeptides involving dialysis on acommercially-useful scale.

It is an object of the present invention to provide a method ofproducing polypeptides, particularly tyrosine kinase receptor-relatedpolypeptides, which provides improved yields compared to prior artprocesses. It is a further object of the present invention to provide amethod which provides a product with improved stability compared toprior art processes.

SUMMARY OF THE INVENTION

According to one aspect of the invention there is provided a method ofproducing tyrosine kinase receptor-related polypeptides, the methodcomprising expressing a tyrosine kinase receptor-related polypeptide ina recombinant expression system and separating expressed monomerictyrosine kinase receptor-related polypeptide from multimeric form(s) ofthe expressed polypeptide in a separation step, the separation stepallowing refolding of the expressed tyrosine kinase receptor-relatedpolypeptide into a biologically active form.

The method is advantageous over known processes for several reasons.First, the method produces significantly higher yields than knownprocesses. Second, the method is scalable allowing production ofpolypeptide at commercially useful levels. Third, the method does notrequire a separate dialysis-based refolding step. Dialysis requireslarge amounts of expensive dialysis buffer since it requires refoldingat low polypeptide concentrations, and a lengthy period of time forrefolding. Methods involving a dialysis step require a recovery step forcapture of the polypeptide. This can be done for instance by ionexchange or affinity separation. The use of ion exchange requiresincreased levels of NaCl to elute product; this is disadvantageous inthat it causes further aggregation of the polypeptide. The use ofaffinity separation for example using a His tag on a nickel chelatingcolumn also requires relatively high NaCl levels and elution withimidazole requires gel filtration to remove. Fourth, the process is muchquicker than prior art processes involving dialysis, which is usuallydone overnight. Fifth, the product of the method is more stable. Ratherthan having to be snap frozen immediately after production, it can bekept normally refrigerated (at about 4° C.) and is biologically activefor at least three months. As the product has lower dimer levels thereis less tendency for aggregation seeded by dimers to take place. Sixth,the product can be produced at higher concentrations (up to 650 μM)without strand swap dimers being produced. Seventh, as the polypeptideproduct is not in contact with urea for the lengthy periods requiredduring a dialysis procedure it is less likely to be amidated. Amidationcan affect biological activity. It may also make it more difficult tocouple the polypeptide to matrices using amine coupling methods. Thismakes products of the method of the present invention more useful incertain applications such as biosensors.

The tyrosine kinase receptor may be native TrkA, TrkB, TrkC; or abiologically active homologue, variant, portion of those receptors or aconstruct including a homologue, variant, or portion thereof. Preferablythe polypeptide is selected from TrkAIg₂ and TrkBIg₂. Particularlypreferred polypeptides for production by the method of the presentinvention are the Ig₂ subdomains of the TrkA, TrkB, and TrkC receptors.Most preferably the polypeptide is TrkAIg₂ or TrkAIg_(2.6).

Preferred constructs may include additional C terminal sequence from thecorresponding native receptor.

The polypeptide may be expressed with a histidine tag sequence. Thetyrosine kinase sequence is preferably human. The tyrosine kinasereceptor-related polypeptide may be expressed in insoluble form.Preferably the tyrosine kinase receptor-related polypeptide is expressedin bacterial inclusion bodies. The multimeric forms of the polypeptidemay include dimers. The polypeptide is preferably able to bind a ligandof the corresponding native tyrosine kinase receptor with high affinity.

The separation step preferably involves gel filtration. The separationstep is preferably carried out at a salt concentration between 0 mM and500 mM, and more preferably above 25 mM and below 200 mM, mostpreferably at a salt concentration of about 100 mM, for example in therange 80 mM to 120 mM. The gel used in the gel filtration step ispreferably able to separate molecules having a molecular weight of about12 to 40 kDa The gel may be for example Sephacryl 200, SuperDex 75 orSuperDex 200.

The separation step is preferably carried out at an alkaline pH.Preferably, the separation step is carried out at a pH below one wheredenaturation occurs. For example, the step may be carried out attypically between pH 8 and 9. Most preferably, the filtration step iscarried out at about pH 8.5. This is unexpected in the case of TrkA asTrkAIg₂ has a calculated P_(i) of between 4.6 and 6.0 dependant on theprogram used for the calculation.

TrkAIg₂ has a high level of β sheet and most proteins like thisaggregate and precipitate near their pI. TrkAIg₂, however, precipitatesand aggregates at pH's around physiological pH, and significantlydifferent from its pI and at salt concentrations that normally maintainsuch proteins in solution.

In a preferred arrangement, polypeptide is eluted from the gelfiltration step at a flow rate of about 2.5 ml/min, and monomer iscollected after about 93 minutes. This will, however, vary according tothe apparatus and conditions under which it is operated. Preferably, thepolypeptide is produced in a bacteria-based expression system.

According to a preferred aspect of the invention there is provided amethod of purifying recombinant TrkAIg₂ or TrkAIg_(2.6) from inclusionbodies in a bacterial expression system in which monomeric TrkAIg₂ isseparated from a mixture including monomeric and multimeric TrkAIg₂ by agel filtration step and allowed to refold into a biologically activeform. Typically, the multimeric TrkAIg₂ will comprise dimeric TrkAIg₂.

The invention also provides a stable preparation of TrkAIg₂ obtained, orobtainable, by a method according to the invention and comprising lessthan 20% of TrkAIg₂ dimer or dimer aggregate, more preferably less than1% of TrkAIg₂ dimer or dimer aggregate, most preferably less than 0.1%of TrkAIg₂ dimer or dimer aggregate.

The invention also provides a stable preparation of TrkAIg₂ obtained, orobtainable, by a method according to the invention and comprising morethan 80% TrkAIg₂ monomer, more preferably more than 99% TrkAIg₂ monomer,most preferably 100% TrkAIg₂ monomer. Preferably, the monomer issubstantially all in a biologically active form.

The invention also provides a preparation of TrkAIg_(2.6) obtained, orobtainable, by a method according to the invention and comprising lessthan 20% of TrkAIg_(2.6) dimer or dimer aggregate, more preferably lessthan 1% of TrkAIg_(2.6) dimer or dimer aggregate, most preferably lessthan 0.1% of TrkAIg₂ dimer or dimer aggregate.

The invention also provides a stable preparation of TrkAIg_(2.6)obtained or obtainable, by a method according to the invention andcomprising more than 80% TrkAIg_(2.6) monomer, more preferably more than99% TrkAIg_(2.6) monomer, most preferably 100% TrkAIg_(2.6) monomer.Preferably, the monomer is substantially all in a biologically activeform.

The invention also provides a stable preparation of TrkBIg₂ obtained, orobtainable, by a method according to the invention comprising less than20% of TrkBIg₂ dimer or dimer aggregate, more preferably less than 1% ofTrkBIg₂ dimer or dimer aggregate, most preferably less than 0.1% ofTrkBIg₂ dimer or dimer aggregate.

The invention also provides a stable preparation of TrkBIg₂ obtained, orobtainable, by a method according to the invention comprising more than80% TrkBIg₂ monomer, more preferably more than 99% TrkBIg₂ monomer, mostpreferably 100% TrkBIg₂ monomer. Preferably, the monomer issubstantially all in a biologically active form.

The invention also provides a stable preparation of TrkCIg₂ obtained, orobtainable, by a method according to the invention and comprising lessthan 20% of TrkCIg₂ dimer or dimer aggregate, more preferably less than1% of TrkCIg₂ dimer or dimer aggregate, most preferably less than 0.1%of TrkCIg₂ dimer or dimer aggregate.

The invention also provides a stable preparation of TrkCIg₂ obtained, orobtainable, by a method according to the invention and comprising morethan 80% TrkCIg₂ monomer, more preferably more than 99% TrkCIg₂ monomer,most preferably 100% TrkCIg₂ monomer. Preferably, the monomer issubstantially all in a biologically active form.

According to another aspect of the invention there is provided a methodof producing immunoglobulin-like polypeptide monomers from a mixture ofmonomeric and multimeric forms of the polypeptide, the method comprisingexpressing the polypeptide in a recombinant expression system andseparating polypeptide monomers from multimeric forms of the polypeptidein a separation step, the separation step allowing the polypeptide torefold to a biologically active form. Thus the invention provides amethod of purifying immunoglobulin-like polypeptides which has some orall of the advantages described above. The separation step preferablyincludes gel filtration.

BRIEF DESCRIPTION OF THE DRAWINGS

Methods and products in accordance with the invention will now bedescribed, by way of example only, with reference to the furtheraccompanying FIGS. 2 to 22 in which:

FIG. 2 shows the amino acid sequences of (A) TrkAIg₂ and TrkAIg_(2.6);(B) TrkBIg₂ truncated and full length forms (in bold; pET15b sequences(MGSSHHHHHH SSGLVPRGSHM) in unbolded form); and (C) TrkCIg₂ truncatedand full length forms (in bold; pET15b sequences (MGSSHHHHHHSSGLVPRGSHM) in unbolded form);

FIG. 3 is a overlap of traces from an FPLC machine illustratingcomparative experiments with a prior art dialysis method and a method inaccordance with the invention;

FIG. 4 is a series of traces illustrating the results of experiments inwhich pH was altered;

FIG. 5 is a series of traces illustrating comparative experiments withvolume of dialysis buffer;

FIG. 6 shows results of mass spectrometry experiments on TrkAIg₂6His andTrkAIg_(2.6)6His produced by the invention;

FIG. 7 illustrates the results of binding activity studies forTrkBIg₂6His, with A: BDNF and B: NT4;

FIG. 8 illustrates the results of binding activity studies withTrkAIg₂6His with NGF;

FIG. 9 illustrates the results of binding activity studies withTrkAIg_(2.6)6His with NGF;

FIG. 10 shows results of mass spectrometry experiments on TrkBIg₂6Hisproduced by the invention;

FIG. 11 shows results of PC12 cell neurite outgrowth bioassay usingTrkAIg₂6His;

FIG. 12 shows result of mass spectrometry experiments on TrkCIg₂6Hisproduced by the invention;

FIG. 13 illustrates the results of binding activity studies withTrkCIg₂6His with NT-3;

FIG. 14 illustrates the predicted mRNA structure of TrkAIg₂6His;

FIG. 15 illustrates the predicted mRNA structure of TrkAIg₂ noHis;

FIG. 16 illustrates an example of mutations required to facilitateexpression of TrkAIg₂noHis;

FIG. 17 illustrates the predicted mRNA structure for the mutant sequenceshown in FIG. 16;

FIG. 18 shows an SDS-PAGE gel showing cell extracts from E. coliexpressing the pET24a-TrkAIg₂ noHis mutant sequence shown in FIG. 16;

FIG. 19 shows results of mass spectrometry experiments on TrkAIg₂ noHisproduced by the invention;

FIG. 20 shows results of PC12 cell neurite outgrowth bioassay usingTrkAIg₂ noHis;

FIG. 21 illustrates examples of mutations required to facilitateexpression of TrkBIg₂noHis; and

FIG. 22 illustrates examples of mutations required to facilitateexpression of TrkCIg₂noHis.

Definitions:

“Polypeptide”: This term embraces proteins i.e. naturally occurring fulllength biologically active polypeptides.

“TrkAIg₂”: is a polypeptide having the amino acid sequence shown in boldin FIG. 2A (whether with or without the additional six amino acidresidues underlined which lead to the variant TrkAIg_(2.6)) andhomologues (for example as a result of conservative substitutions of oneor more amino acid residues in the sequence) or variants includingsequences to enhance expression and/or purification such as the his tagand thrombin cleavage sequence shown in unbolded form in FIG. 2A.

“TrkBIg₂” and “TrkCIg₂” have corresponding meanings with reference tothe sequences shown in FIGS. 2B and 2C respectively.

“TrkAIg₂6His” represents variants including the His tag and “TrkAIg₂noHis” represents variants not including the His tag. Similar termsapply to corresponding variants of TrkB and TrkC and proteins thereof.

Specific Description

Production of Histidine-Tagged TrkIgs

Production of Recombinant TrkAIg₂6His and TrkAIg_(2.6)6His Polypeptide

Recombinant TrkAIg₂6His was produced in E. coli BL21 (DE3) cells usingthe method described in WO99/53055 in the section headed “Expression ofTrkAIg_(1,2), TrkAIg₁ and TrkAIg₂” and incorporating a 6-histidine tagto the N-terminus of the polypeptide as shown in FIG. 2A. RecombinantTrkAIg_(2.6) 6His was prepared in a similar manner.

Purification and Refolding of TrkAIg₂6His Polypeptide

The harvested cells were resuspended in 10% glycerol, frozen at −70° C.in liquid nitrogen and the resulting pellet was passed three timesthrough an XPress (AB Biox). The extract was centrifuged at 10,000 rpm,4° C. for 30 min to pellet the insoluble inclusion bodies containing therecombinant polypeptide.

The inclusion bodies were washed in 500 ml 1% (v/v) Triton X-100, 10 mMTris HCl pH8.0, 1 mM EDTA followed by 500 ml 1M NaCl, 10 mM Tris HCl pH8.0, 1 mM EDTA and finally 10 mM Tris HCl pH8.0, 1 mM EDTA.

The inclusion bodies were then solubilised in 20 mM Na Phosphate, 30 mMImidazole, 8M Urea (pH 7.4) and clarified by centrifugation. 6MGuanidinium may also be used in place of 8M urea throughout.

The resulting mixture was loaded on a 5 ml HisTrap column(Pharmacia),and washed with 50 ml 20 mM NaPhosphate, 30 mM Imidazole, 8M Urea pH7.4. The purified TrkAIg₂6His was eluted with 25 ml 20 mM NaPhosphate,300 mM Imidazole, 8M Urea (pH7.4).

In order to allow the purified recombinant TrkAIg₂6His polypeptide torefold, the eluant of the previous step was then applied to a SuperDex200 gel filtration column, and equilibrated in 20 mM NaPhosphate, 100 mMNaCl, at pH 8.5. The column had a height of 65 cm, width 2.6 cm andvolume of 345 ml when pre-packed by the manufacturer. The flow rate atmaximum pressure was 2.5 ml/min.

Any aggregate (i.e. dimer aggregates) eluted in the void volume. Dimerwas eluted after about 80 min and monomer eluted after about 93 min. Theelution time of a protein will be dependent on dimensions of column andis dependent on its size. This is described by the following formula:R=VO/Vewhere R is retention coefficient of a protein, Ve is the volume at whichthe protein is eluted, VO is void volumewhere Ve=232.5 ml VO is 122 ml122/232.5=0.53

TrkAIg₂6His monomer on a SuperDex 200 gel has a retention coefficient of0.53.

By way of comparison the polypeptide was also folded by dialysis firstagainst 20 mM Tris HCl, 50 mM NaCl, pH 8.5, recaptured on a His Trapcolumn and eluted with 25 ml 20 mM NaPhosphate, 300 mM Imidazole, 8MUrea pH 7.4.

Purification traces from the Biocad Sprint FPLC (Biocad) which showelution of monomer, dimer and aggregates of TrkAIg₂6His under variousconditions were prepared. FIG. 3 shows an overlay trace comparingelution of TrkAIg₂6His with refolding by dialysis (“Dialysis”) and withrefolding on a column in a method according to the invention(“SuperDex”). It will be seen that the method of the invention produceshigher levels of monomer compared to the prior art process.

The splice variant TrkAIg_(2.6)6His was prepared and purified in asimilar manner.

Effect of pH on Elution of TrkAIg₂6His

TrkAIg₂6His was expressed in E. coli as described above. Purifiedinclusion bodies were solubilised in 20 mM Na Phosphate, 30 mMImidazole, 8M Urea pH 7.4 and clarified by centrifugation. The resultingmutant was affinity purified on HisTrap column and eluted with 25 ml 20mM NaPhosphate, 300 mM Imidazole, 8M Urea pH 7.4. The eluted TrkAIg₂6Hiswas applied to SuperDex 200 gel filtration column (Pharmacia) andequilibrated in 20 mM NaPhosphate, 100 mM NaCl, pH 8.5. The flow ratewas 2.5 ml/min. The time taken for elution is determined by size ofprotein. Peaks of monomer, dimer and aggregate are indicated atapproximately 93 minutes, 80 minutes and 50 minutes respectively. Theresults are given in FIG. 4 whichshows the results of elution at pH7.4,8.0, 8.5 and 9.0. The results indicate that pH 8.5 was best with thegreatest yield, lowest amount of aggregate, and highest levels ofmonomer.

Comparative Example: Separation of TrkAIg₂6His Monomer, Dimer andAggregate with Refold by Dialysis. Amount of Dialysis Buffer Required.Subsequent Analysis by Elution from SuperDex 200.

TrkAIg₂6His was expressed in E. coli. Purified inclusion bodies weresolubilised in 20 mM NaPhosphate, 30 mM Imidazole, 8M Urea pH 7.4 andclarified by centrifugation. The solution was affinity purified onHisTrap column and eluted with 25 ml 20 mM NaPhosphate, 300 mMImidazole, 8M Urea pH 7.4. TrkAIg₂6His was folded by dialysis (using 1litre, 2 litres or 4 litres) overnight against 20 mM Tris HCl, 50 mMNaCl, pH 8.5, recaptured on a HisTrap column and eluted with 25 ml 20 mMNaPhosphate, 50 mM EDTA, 8M Urea pH 7.4. Final analysis was using aSuperDex 200 gel filtration column equilibrated in 20 mM NaPhosphate,100 mM NaCl, pH 8.5. Flow rate was 2.5 ml/min. 2-4 litres were requiredfor washing. 4 litres gave the highest yield of monomer. This shows howlarge volumes of buffer are needed if dialysis is to be used forrefolding of the expressed polypeptide.

Results are shown in FIG. 5.

Characterisation of TrkAIg₂6His and TrkAIg_(2.6)6His Produced by Methodof the Invention

The expressed TrkAIg₂6His (A) and TrkAIg_(2.6)6His (B) polypeptides weresubjected to MALDITOF mass spectrometry and the results are shown inFIG. 6. The molecular mass of the polypeptides was determined using a PEBiosystems Voyager-DE STR Matrix-Assisted Laser DesorptionTime-of-Flight (MALDITOF) mass spectrometer with a nitrogen laseroperating at 337 nm. The matrix solution was freshly prepared sinapinicacid at a concentration of 1 mg/1001 μl in a 50:50 mixture ofacetonitrile and 0.1% trifluoroacetic acid. 0.51 μl of sample and matrixwere spotted onto the sample plate. The sample was calibrated againstCalmix 3 (PE Biosystems) run as a close external standard. The spectrumwas acquired over the range 5000-80,000 Da, under linear conditions withan accelerating voltage of 25,000 V, an extraction time of 750 nsecs andlaser intensity of 2700.

The molecular weight of TrkAIg₂6His was found to be 15,717.96 Da. Thisis almost exactly as predicted by theoretical calculation of themolecular weight (15716.3 Da, after loss of the N-terminal methionine,which we have previously found to be removed in proteins incorporatingthe 6 histidine tag from the expression vector pET15b).

The molecular weight of TrkAIg_(2.6)6His was found to be 16,575.3 Da.This is almost exactly as predicted by theoretical calculation of themolecular weight (16,574.4 Da).

Stability

TrkAIg₂6His and TrkAIg_(2.6)6His produced as described above hasremained stable when kept at 4° C. for three months and has retained itsbiological activity.

Improvements in stability may be achieved using conventional additivessuch as glycerol.

Biological Activity of TrkAIg₂6His Produced by the Method of theInvention

i Guinea Pig Hind Limb Pain Responses

Biological activity of TrkAIg₂6His produced by the method of theinvention was tested in guinea pigs (Djouhri, L. et al (2001) JNeuroscience 21 p8722-8733). CFA (Complete Freund's Adjuvant) wasinjected into the hind limb and knee of guinea pigs. This causesinflammation which leads to an increase of NGF levels. This makes theanimal more susceptible to feeling pain. Intracellular recordings weremade from the cell bodies of L6 (lumbar), and S1 (sacral) DRG neuronswith glass microelectrodes and action potentials were evoked bystimulation of DRG with a pair of platinum electrodes. The recordingswere made 1, 2 and 4 days after CFA administration. The C and Aδ fibresare nociceptive —they transmit pain signals to the brain. α and β fibresdo not. Spontaneous firing of nociceptive neurons without outsidestimulation is thought to be responsible for inflammatory andneuropathic pain in humans.

TrkAIg₂6His was injected on days 2, 3 and 4 with 0.45 μg into hind limband knee on guinea pig. Adding TrkAIg₂6His, which sequesters theendogenous NGF, abolished CFA-induced increases in following frequencyand in spontaneous firing. This meant complete cessation of abnormalpain.

TrkAIg₂6His was therefore able to inhibit pain response in CFA inducedpain fibre firing in guinea pigs.

ii PC12 Cell Bioassays

Biological activity of TrkAIg₂6His produced by the method of theinvention was tested by PC12 neurite outgrowth bioassay. PC12 cells area rat phaeochromocytoma cell line which grow neurites in response to thepresence of NGF, which binds receptors present on the cell surface. PC12cells were plated out at 2×10⁴ cells per well in complete DMEM medium(including 100 units/ml penicillin, 100 μg/ml streptomycin, 10% horseserum, 10% Foetal Calf Serum (FCS) and 2 mM glutamine) oncollagen-coated 24-well plates. NGF was added at 1 ng/ml and TrkAIg₂6Hiswas added at varying concentrations. Results are shown in FIG. 11,photographs of neurite outgrowth after 48 hours. Cells were fixed beforephotographing. FIG. 11A shows neurite outgrowth with 1 ng/ml NGF andFIG. 11B shows no neurite outgrowth when 1.25 μm TrkAIg₂6His is added.

TrkAIg₂6His was therefore able to prevent neurite growth in response toNGF in the PC12 cell line.

Sub-Cloning of the TrkBIg₂6His Domain

The TrkBIg₂6His protein comprises residues 286 to 430 of the matureprotein, and has a further 21 residues at the NH₂ terminus whichconstitute the histidine expression tag and associated thrombin cleavagesequence. cDNA coding for the TrkBIg₂₆His domain was PCR amplified fromλZAP-pBluescriptllSK⁽⁻⁾/TrkB, a non-catalytic form of human TrkB clonedby us (Allen et al (1994) Neuroscience 60 p825-834). Primers (MWGBiotech) incorporated a Nde1 site in the forward primer(CGCATATGGCACCAACTATCACATTTCTCGAATCTC), and a BamHI site in the reverseprimer:

(GCGGATCCCTATTAATGRRCCCGACCGGTTTTATC).

The PCR product was subcloned into pET15b (Novagen), using Nde1 andBamHI sites, to create the expression vector pET15b-TrkBIg₂6His.

A truncated version of TrkBIg₂6His, shown in FIG. 2B, was also producedin exactly the same way but using the amino acids 286-383. This form wasco-crystallised with its ligand NT4 and an X-ray crystal structuresolved.

Production of Recombinant TrkBIg₂6His Polypeptide

Electro-competent E. coli BL21 (DE3) cells were transformed withpET15b-TrkBIg₂, and expression was carried out in accordance with thepET (Novagen) manual. After transformation, E. coli cell lysates wereanalysed by SDS-PAGE for expression of the 18.5 kDa protein. TrkBIg₂6Hisprotein was expressed at high levels in the urea-soluble fraction, butnot in the other fractions. 2 ml of 2YT broth (containing 200 μg/1 mlampicillin) was inoculated with a colony and grown at 37° C. to mid logphase. This was used to inoculate 50 ml of 2YT broth (containing 200μg/ml ampicillin), which was grown at 37° C. to mid log phase. This wasused to inoculate 5 litres of 2YT broth (containing 200 μg/mlampicillin), which was grown to an optical density of 1.0 at 600 nm. 1mM IPTG was added to induce protein expression and cells were grownovernight at 37° C. The harvested cells were resuspended in 10% glyceroland frozen at −80° C. (8 pellets). Pellets were lysed by passing 3 timesthrough an Xpress, then washed with 20 mM sodium phosphate buffers (pH8.5) containing, in succession, 0.1M NaCl, 1% Triton X-100, and finally1M NaCl. This removed all soluble matter, leaving inclusion bodiescontaining insoluble protein.

Refolding of TrkBIg₂6His Polypeptide

Insoluble TrkBIg₂6His protein contained in the inclusion bodies wasreleased from the cells with an Xpress, and washed to remove solublematter. The purified inclusion bodies were solubilised in 8M urea buffer(20 mM sodium phosphate, pH 8.5, 1 mM β-mercaptoethanol), with a“Complete” proteinase inhibitor cocktail tablet (Roche) and incubated atroom temperature for 2 hours with gentle shaking. 6M Guanidinium may besubstituted for 8M urea. TrkBIg₂6His protein was purified on a HisTrapnickel column (Pharmacia), under reducing conditions (20 mM sodiumphosphate, pH 8.5, 8M urea, 10 mM imidazole), and eluted using 300 mMimidazole. Refolding took place under non-reducing conditions (20 mMsodium phosphate, pH 8.5, 100 mM NaCl) on a SuperDex 200 gel-filtrationcolumn (Pharmacia). Fractions from the peak corresponding to a molecularweight of approximately 18.5 kDa were pooled; these containedTrkBIg₂6His monomer.

Characterisation of TrkBIg₂6His Produced by Method of the Invention

The molecular mass of TrkBIg₂6His was determined using a PE BiosystemsVoyager-DE STR MALDITOF mass spectrometer, with a nitrogen laseroperating at 337 nm. The matrix solution was freshly prepared sinapinicacid at a concentration of 1 mg/100 μl in a 50:50 mixture ofacetonitrile and 0.1% trifluoroacetic acid. 0.5 μl of sample and matrixwere spotted onto the sample plate. The sample was calibrated againstCalmix 3 (PE Biosystems) run as a close external standard. The spectrumwas acquired over the range 5000-80,000 Da, under linear conditions withan accelerating voltage of 25,000 V, an extraction time of 750 ns and alaser intensity of 2700. Results are shown in FIG. 10.

The molecular weight of TrkBIg₂₆His was found to be 18,451.7 Da This isalmost exactly as predicted by theoretical calculation of the molecularweight (18,449.1 Da).

Sub-Cloning of Recombinant TrkCIg₆His Domain

The TrkCIg₂6His protein comprises residues 300 to 399 of the matureprotein, and has a further 21 residues at the NH₂ terminus whichconstitute the histidine expression tag and associated thrombin cleavagesequence. cDNA coding for the TrkCIg₂6His domain was PCR amplified usinga forward primer which incorporated a Nde1 site(CGCATATGACTGTCTACTATCCCCCAC) and a reverse primer which incorporated aBamH1 site (GCGGATCCTTATCAGGGCTCCTTGAGGAAGTGGC). The PCR product wassubcloned into pET15b (Novagen) using Nde1 and BamH1 restriction sites,to create the expression vector pET15b-TrkCIg₂6His.

Production of Recombinant TrkCIg₂₆His Polypeptide

Electrocompetent E. coli BL21 (DE3) cells were transformed withpET15b-TrkCIg₂6His and expression was carried out in accordance with pET(Novagen) manual. After transformation E. coli lysates were anaylsed bySDS-PAGE for expression of the 13.8 kDa protein. TrkCIg₂6His protein wasexpressed at high levels in the urea-soluble fraction but not in otherfractions. 2 ml of 2YT broth (containing 200 μg/ml ampicillin), wasinoculated with a colony which was grown at 37° C. to mid log phase.This was used to inoculate 50 ml of 2YT broth (containing 200 μg/mlamplicillin) which was grown at 37° C. to mid log phase. This was usedto inoculate 5 litres of 2YT broth (containing 200 μg/ml ampicillin),which was grown to an optical density of 1.0 at 600 nm. 1 mM IPTG wasadded to induce protein expression and cells were grown overnight at 37°C. The harvested cells were resuspended in 10% glycerol and frozen at−80° C. (8 pellets). Pellets were lysed by passing 3 times through anXpress, and then washed with 20 mM sodium phosphate buffer (pH8.0)containing, in succession, 0.1M NaCl, 1% Triton X-100, and finally 1MNaCl. This removed all soluble matter, leaving inclusion bodiescontaining insoluble protein. All washes were at 4° C.

Refolding of TrkCIg₂6His Polypeptide

Insoluble TrkCIg₂6His protein contained in the inclusion bodies wasreleased from the cells with Xpress, and washed to remove solublematter. The purified inclusion bodies were solubilised in 8M urea buffer(20 mM sodium phosphate pH 8.0, 1 mM β-mercaptoethanol) and incubated atroom temperature for 2 hours with gentle shaking. 6M Guanidinium may besubstituted for 8M urea. TrkCIg₂₆His protein was purified on a HisTrapnickel column (Pharmacia) in 20 mM sodium phosphate, pH 8.0, 8M urea, 10mM imidazole, 1 mM β-mercaptoethanol and eluted using 300 mM imidazole.Refolding was in 20 mM sodium phosphate, pH 8.0, 100 mM NaCl, 1 mMβ-mercaptoethanol on a SuperDex 200 gel filtration column (Pharmacia).Fractions from the peak corresponding to a molecular weight ofapproximately 13.8 kDa were pooled. These contained TrkCIg₂6His monomer.The retention coefficient of TrkCIg₂6His is 0.51.

Characterisation of TrkCIg₆His Produced by Method of the Invention

The molecular mass of TrkCIg₂6His was determined using a PE Biosystemsvoyager-DE STR MALDITOF mass spectrometer, with a nitrogen laserseparating at 337 nm. The matrix solution was freshly prepared sinapinicacid at a concentration of 1 mg/100 μl in a 50:50 mixture ofacetonitrile at 0.1% trifluoracetic acid. 0.5 μl of sample and matrixwere spotted onto the sample plate. The sample was calibrated againstCalmix 3 (PE Biosystems) run as a close external standard. The spectrumwas acquired over the range 5000-80,000 Da, under linear conditions withan accelerating voltage of 25,000 V, an extraction time of 750 ns and alaser intensity of 2700. Results are shown in FIG. 12.

The molecular weight of TrkCIg₂6His was found to be 13,681.9 Da. This isalmost exactly as predicted by a theoretical calculation of themolecular weight (13,685.3 Da) taking into account loss of theN-terminal methionine, which we have previously found to be removed inproteins incorporating the 6 histidine tag from the expression vectorpET15b.

Activity Studies: Binding Activity of TrkAIg₂6His, TrkAIg_(2.6)6His,TrkBIg₂6His and TrkCIg₂6His

The resulting monomeric recombinant TrkIg₂ were shown to bind thenatural ligands of the respective full length receptors with similaraffinity to the wild type receptor i.e. this may be expected to bebiologically active. In contrast strand swapped dimeric TrkBIg₂6Hiswould be biologically inactive.

The ability of TrkIg₂ domains to bind to their respective ligands wasmeasured using plasmon surface resonance with a BiaCore system(BiaCore). TrkIg₂ was bound to the matrix of a CM5 chip by aminecoupling.

Binding Activity of TrkIgs. Surface Plasmon Resonance

i. TrkBIg₂₆His

BDNF was passed over the chip at 0.1-25 nM. Association and dissociationrates were estimated according to a 1:1 Langmuir binding model, giving aKD of 790 pM. NT-4 was passed over the chip at 1-100 nM. Association anddissociation rates were estimated according to a 1:1 Langmuir bindingmodel, giving a KD of 260 pM. Results are shown in FIG. 7. FIG. 7A showsthe results of experiments with BDNF at 0.1, 0.2, 0.5, 1, 2, 5, 10 and25 nM (all duplicate). Association and dissociation were fitted to a 1:1Langmuir model, giving a KD of 790 pM (Chi²=4.39).

FIG. 7B shows the results of experiments with NT-4 at 1, 5, 25, 50, 75and 100 nM (all duplicate). Association and dissociation were fitted toa 1:1 Langmuir model, giving a KD of 260 pM (Chi²=2.85).

ii. TrkAIg₂6His

NGF was passed over the chip at 0.1-100 nM. Association and dissociationrates were estimated according to a 1:1 Langmuir binding model, giving aKD of 92.6 pM. The results are shown in FIG. 8.

iii. TrkAIg_(2.6)6His

NGF was passed over the chip at 0.1-100 nM. Association and dissociationrates were estimated according to a 1:1 Langmuir binding model, giving aKD of 79.2 pM. Results are shown in FIG. 9. This is a very high affinityand commensurate with known characteristics of the biological membranebound wild type receptor.

Djouhri, L. et al (supra) indicates TrkAIg₂6His is active in vivo toprevent abnormal fibre firing of noiceptive neurons.

iv. TrkCIg₂6His

NT-3 was passed over the chip at 0.1-100 nM. Regeneration was with 10 μl10 mM glycine, pH 1.5. Association and dissociation rates were estimatedaccording to a 1:1 Langmuir binding model, giving KD of 200 μm. Theresults are shown in FIG. 13.

Production of Non-Histidine-Tagged TrkAIgs

Cloning of TrkAIg₂ noHis

TrkAIg₂ was cloned into pET24a for the expression of TrkAIg₂ without thehistidine tag (TrkAIg₂ noHis). Without modification this does notexpress protein.

It is known that secondary structure in the mRNA transcript caninterfere with the AUG translation initiation codon and/or the ribosomebinding site. Using the software MFOLD(http://bioweb.pasteur.fr/seqanal/interfaces/mfold.html) to investigatethe secondary structure it was seen that the transcription start sitewas not ideal for expression. FIG. 14 shows the predicted mRNA structurefor TrkAIg₂6His in pET15b. The mRNA coding for the 6His tag is outlined,as is the ribosome binding site (RBS) and the codon for a prolineresidue (PRO). FIG. 15 shows the predicted mRNA structure of TrkAIg₂noHis in pET24a. It can be seen that the initiation site is much lessaccessible than in the 6His version. Similar restrictions also arise inpredicted structures of TrkBIg₂noHis and TrkCIg₂noHis.

Using computer software to predict the resulting mRNA structures,various silent mutations were introduced into the DNA structure ofTrkAIg₂ noHis to allow access to the RBS. FIG. 16 shows an example of aresulting DNA sequence, compared with the wild-type. Mutated bases aremarked bold. The resulting mRNA-structure predicted by MFOLD is shown inFIG. 17. Examples of suitable mutated sequences for TrkBIg₂noHis areshown in FIG. 21 and for TrkCIg₂noHis in FIG. 22.

TrkAIg₂ was amplified by PCR from the pET15b-TrkAIg₂6His plasmid usingthe forward primer GGAATTCCATATGCCTGCTTCAGTACAATTACACACGGCGGTC whichincorporates mutated bases and reverse primerCCGCTCGAGTTATCATTCGTCCTTCTTCTCCACCGGGTCTCCA. Primers include sites forNdeI and XhoI respectively at the 5′ and 3′ of TrkAIg₂noHis. Between100-1000 pmol primers were used per reaction.

Hot start PCR was carried out over 30 cycles in a thermal cycler. Afteran initial denaturing temperature of 94° C. for 15 minutes, PFUpolymerase was added and 30 cycles of denaturation at 94° C. for 1minute, annealing at 67° C. for 1 minute and extension at 72° C. for 1minute were carried out. Final extension was 10 minutes at 72° C.followed by a 4° C. holding step. PCR products were analysed by agarosegel electrophoresis TrkAIg₂noHis mutants were subcloned into NdeI andXhoI digested pET24a to create the expression vector pET24a-TrkAIg₂noHis.

FIG. 18 shows SDS-PAGE analysis of TrkAIg₂ noHis expressed in E. coli;(M) markers, (W) whole cell extract, (S) soluble extract, (1) insolubleextract. It can be seen that TrkAIg₂noHis is expressed mainly in theinsoluble fraction.

Production of Recombinant TrkAIg₂ noHis Polypeptide

Electrocompetent E. coli BL21 (DE3) cells were transformed withpET24a-TrkAIg₂ noHis and expression was carried out in accordance withthe pET (Novagen) manual. After transformation E. coli lysates wereanalysed by SDS-PAGE for expression of the 13.5 kDa protein. TrkAIg₂noHis protein was expressed at high levels in the urea-soluble fractionbut not in other fractions. 2 ml of 2YT broth (containing 50 μg/mlkanomycin), was inoculated with a colony which was grown at 37° C. tomid log phase. This was used to inoculate 50 ml of 2YT broth (containing50 μg/ml kanomycin), which was grown at 37° C. to mid log phase. Thiswas used to inoculate 5′ litres of 2YT broth (containing 50 μg/mlkanomycin) which was grown to an optical density of 1.0 at 600 nm. 1 mMIPTG was added to induce protein expression and cells were grown for 3hours at 37° C. The harvested cells were resuspended in 10% glycerol andfrozen at −80° C. (8 pellets).

Inclusion Body Preparation

Pressed cells were mixed in 20 mM Tris buffer pH 8.5 1 mM PMSF, 10 mMEDTA, gently pipetted and 20 mM Tris buffer pH 8.5 added. These werecentrifuged at 9000 rpm for 60 minutes, and supernatant removed. Theprocedure was repeated with 20 mM Tris buffer pH 8.5, 1 mM PMSF, 10 mMEDTA and 1M NaCl, added and then with 1% Triton X-100 added. Then afinal wash was carried out with 20 mM Tris buffer pH 8.5, 1 mM PMSF, 10mM EDTA. This was subsequently centrifuged at 9000 rpm for 30 minutes.Supernatant was removed. All washes were at 4° C. Inclusion bodies werefrozen at −70° C.

Inclusion bodies were solubilised in 8M urea in 20 mM Tris buffer pH 8.5with 25 mM DTT added for three hours at 14° C.

Refolding of TrkAIg₂ noHis Polypeptide

Insoluble TrkAIg₂ noHis protein contained in the inclusion bodies wasreleased from the cells with an Xpress, and washed with salt and TritonX100 to remove soluble matter. The purified inclusion bodies weresolubilised in 8M urea buffer (20 mM Tris pH 8.5, 25 mM DTT) andincubated at room temperature for 3 hours with gentle shaking.

Purification was carried out using an anion exchange column, such as QSepharose Fast Flow (Pharmacia), equilibrated and run in 8M urea (pH8.5) with 10 mM DTT added. Protein was eluted with a gradient of NaCl,in which the protein eluted at approximately 180 mM NaCl or a step at200 mM NaCl. Eluted protein was refolded at 1 mg/ml on a gel filtrationcolumn in Tris pH 8.5 with 100 mM NaCl.

Refolding with gel filtration was successful with a variety of gelfiltration media: SuperDex 200, SuperDex 75, Sephacryl HR100, andSephacryl HR200. In this system, TrkAIg₂ noHis ran with a retentioncoefficient of 0.55. Unexpectedly it ran a little faster thananticipated compared with TrkAIg₂6His under the same conditions.Increased monomeric form was observed with extended solubilisation.Additionally, the monomeric peak may be finally put onto a Poros Qcolumn to concentrate the protein concentration.

Characterisation of TrkAIg₂ noHis Produced by Method of the Invention

The molecular mass of TrkAIg₂ noHis was determined using a PE BiosystemsVoyager-DE STR MALDITOF mass spectrometer with a nitrogen laseroperating at 337 nm. The matrix solution was freshly prepared sinapinicacid at a concentration of 100 mg/100 μl in a 50:50 mixture ofacetonitrile and 0.1% trifluoracetic acid. 0.5 μl of the sample andmatrix were spotted onto the sample plate. The sample was calibratedagainst Calmix 3 (PE Biosystems) run as a close external standard. Thespectrum was acquired over the range 5000-80,000 Da, under linearconditions with an accelerating voltage of 25,000V, an extraction timeof 750 nsecs and laser intensity of 2700. Results are shown in FIG. 19.

The molecular weight of TrkAIg₂ noHis was found to be 13,561.2 Da. Thisis almost exactly as predicted by theoretical calculation of themolecular weight (13,553 Da).

Biological activity of TrkAIg₂ noHis produced by the method of theinvention: PC12 cell bioassays

Biological activity of TrkAIg₂ noHis produced by the method of theinvention was tested by PC12 neurite outgrowth bioassay. PC12 cells wereplated out at 2×10⁴ cells per well in complete DMEM medium (including100 units/ml penicillin, 100 μg/ml streptomycin, 10% horse serum, 10%FCS and 2 mM glutamine) on collagen-coated 24-well plates. NGF was addedat 1 ng/ml and TrkAIg₂ noHis was added at varying concentrations.

Results from an experiment using TrkAIg₂ noHis refolded on a SuperDex200 column are shown in FIG. 20. Photographs show neurite outgrowthafter 48 hours. Cells were fixed before photographing. FIG. 20A showsneurite outgrowth with 1 ng/ml NGF; FIG. 20B shows no neurite outgrowthwhen no NGF is added; FIG. 20C shows reduced neurite outgrowth when 2.5μm TrkAIg₂ noHis is added; FIG. 20D shows no neurite outgrowth when 4.5μm TrkAIg₂ noHis is added.

Similar results were obtained using TrkAIg₂ noHis refolded on SuperDex75, Sephacryl HR100 and Sephacryl HR200 columns.

TrkAIg₂noHis was therefore able to prevent neurite growth in response toNGF in the PC12 cell line.

1. A method of producing tyrosine kinase receptor-related polypeptides,the method comprising expressing a tyrosine kinase receptor-relatedpolypeptide in a recombinant expression system and separating expressedmonomeric tyrosine kinase receptor-related polypeptide from multimericform(s) of the expressed polypeptide in a separation step, theseparation step allowing refolding of the expressed tyrosine kinasereceptor-related polypeptide into a biologically active form.
 2. Amethod according to claim 1 in which the tyrosine kinase receptor is anative TrkA, TrkB, or TrkC; or a biologically active homologue, variant,portion of those receptors or a construct including a homologue,variant, or portion thereof.
 3. A method according to claim 2 in which aportion of a tyrosine kinase receptor, or a construct including such aportion, is expressed and in which the portion is selected from the 1 g2domains of the TrkA, TrkB, and TrkC receptors respectively.
 4. A methodaccording to claim 3 in which the polypeptide is selected from TrkAIg₂,TrkAIg_(2.6), TrkBIg₂ and TrkCIg₂.
 5. A method according to claim 4 inwhich the polypeptide is TrkAIg₂ or TrkAIg_(2.6)
 6. A method accordingto claim 1, 2, 3, 4 or 5 in which the polypeptide is expressed with ahistidine tag sequence.
 7. A method according to any one of claims 1 to5 in which the polypeptide is expressed without a histidine tagsequence.
 8. A method according to any one of claims 1 to 7 in which thetyrosine kinase sequence is human.
 9. A method according to anypreceding claim in which the tyrosine kinase receptor-relatedpolypeptide is expressed in insoluble form.
 10. A method according toclaim 9 in which the tyrosine kinase receptor-related polypeptide isexpressed in bacterial inclusion bodies.
 11. A method according to anypreceding claim in which the multimeric forms include dimers.
 12. Amethod according to any preceding claim in which the polypeptide is ableto bind a ligand of the corresponding native tyrosine kinase receptorwith high affinity.
 13. A method according to any preceding claim inwhich the separation step involves gel filtration.
 14. A methodaccording to any preceding claim in which the separation step is carriedout at a salt concentration between and including 0 mM and 500 mM.
 15. Amethod according to any preceding claim in which the separation step iscarried out at a salt concentration above 25 mM and below 200 mM.
 16. Amethod according to claim 15 in which the separation step is carried outat a salt concentration of about 100 mM.
 17. A method according to anyone of claims 13 to 16 in which the gel used in the gel filtration stepis able to separate molecules having a molecular weight of about 12 to40 kDa.
 18. A method according to claim 17 in which the gel is Sephadex200 or SuperDex
 200. 19. A method according to claim 18 in which the gelis SuperDex
 200. 20. A method according to any preceding claim in whichthe separation step is carried out at a pH of between 8 and
 9. 21. Amethod according to claim 20 in which the separation step is carried outat a pH of about 8.5.
 22. A method according to any preceding claim inwhich the polypeptide is produced in bacterial-based expression system.23. A method of purifying recombinant TrkAIg₂ or TrkAIg_(2.6) frominclusion bodies in a bacterial expression system in which monomericTrkAIg₂ or TrkAIg_(2.6) is separated from a mixture including monomericand multimeric TrkAIg₂ or TrkAIg_(2.6) by a gel filtration step andallowed to refold into an active form.
 24. A method of purifyingrecombinant TrkBIg₂ from inclusion bodies in a bacterial expressionsystem in which monomeric TrkBIg₂ is separated from a mixture includingmonomeric and multimeric TrkBIg₂ by a gel filtration step and allowed torefold into an active form.
 25. A method of purifying recombinantTrkCIg₂ from inclusion bodies in a bacterial expression system in whichmonomeric TrkCIg₂ is separated from a mixture including monomeric andmultimeric TrkCIg₂ by a gel filtration step and allowed to refold intoan active form.
 26. A preparation of TrkAIg₂ obtained by a methodaccording to any one of claims 3 to 23 and comprising less than 20%TrkAIg₂ dimer or dimer aggregate.
 27. A preparation of TrkAIg₂ accordingto claim 26 comprising less than 10% TrkAIg₂ dimer or dimer aggregate.28. A preparation of TrkAIg₂ according to claim 27 comprising less than1% TrkAIg₂ dimer or dimer aggregate.
 29. A preparation of TrkAIg₂according to claim 28 comprising less than 0.1% TrkAIg₂ dimer or dimeraggregate.
 30. A preparation of TrkAIg₂ obtained by a method accordingto any one of claims 3 to 23 and comprising more than 80% TrkAIg₂monomer.
 31. A preparation of TrkAIg₂ obtained by a method according toany one of claims 3 to 23 and comprising more than 90% TrkAIg₂ monomer.32. A preparation of TrkAIg₂ obtained by a method according to any oneof claims 3 to 23 and comprising more than 99% TrkAIg₂ monomer.
 33. Apreparation of TrkAIg₂ obtained by a method according to any one ofclaims 3 to 23 and comprising 100% TrkAIg₂ monomer.
 34. A preparation ofTrkAIg_(2.6) obtained by a method according to any one of claims 3 to 23and comprising less than 20% TrkAIg_(2.6) dimer or dimer aggregate. 35.A preparation of TrkAIg_(2.6) according to claim 34 comprising less than10% TrkAIg_(2.6) dimer or dimer aggregate.
 36. A preparation ofTrkAIg_(2.6) according to claim 35 comprising less than 1% ofTrkAIg_(2.6) dimer or dimer aggregate.
 37. A preparation of TrkAIg_(2.6)according to claim 36 comprising less than 0.1% of TrkAIg₂ dimer ordimer aggregate.
 38. A preparation of TrkAIg_(2.6) obtained by a methodaccording to any one of claims 4 to 23 and comprising more than 80%TrkAIg_(2.6) monomer.
 39. A preparation of TrkAIg_(2.6) obtained by amethod according to any one of claims 4 to 23 and comprising more than90% TrkAIg_(2.6) monomer.
 40. A preparation of TrkAIg_(2.6) obtained bya method according to any one of claims 4 to 23 and comprising more than99% TrkAIg_(2.6) monomer.
 41. A preparation of TrkAIg₂ obtained by amethod according to any one of claims 4 to 23 and comprising 100%TrkAIg_(2.6) monomer.
 42. A preparation of TrkBIg₂ obtained by a methodaccording to any one of claims 3, 4, 6 to 22 and 24, and comprising lessthan 20% TrkBIg₂ dimer or dimer aggregate.
 43. A preparation of TrkBIg₂according to claim 42 comprising less than 10% TrkBIg₂ dimer or dimeraggregate.
 44. A preparation of TrkBIg₂ according to claim 43 comprisingless than 1% TrkBIg₂ dimer or dimer aggregate.
 45. A preparation ofTrkBIg₂ according to claim 44 comprising less than 0.1% of TrkBIg₂ dimeror dimer aggregate.
 46. A preparation of TrkBIg₂ obtained by a methodaccording to any one of claim 3, 4, 6, to 22 and 24 and comprising morethan 80% TrkBIg₂ monomer.
 47. A preparation of TrkBIg₂ obtained by amethod according to any one of claims 3, 4, 6 to 22 and 24 andcomprising more than 90% TrkBIg₂ monomer.
 48. A preparation of TrkBIg₂obtained by a method according to any one of claims 3, 4, 6 to 22 and 24and comprising more than 99% TrkBIg₂ monomer.
 49. A preparation ofTrkBIg₂ obtained by a method according to any one of claims 3, 4, 6 to22 and 24 and comprising 100% TrkBIg₂ monomer.
 50. A preparation ofTrkCIg₂ obtained by a method according to any one of claims 3, 4, 6 to22 and 25 and comprising less than 20% TrkCIg₂ dimer or dimer aggregate.51. A preparation of TrkCIg₂ according to claim 50 comprising less than10% TrkCIg₂ dimer or dimer aggregate.
 52. A preparation of TrkCIg₂according to claim 51 comprising less than 1% TrkCIg₂ dimer or dimeraggregate.
 53. A preparation of TrkCIg₂ according to claim 52 comprisingless than 0.1% TrkCIg₂ dimer or dimer aggregate.
 54. A preparation ofTrkCIg₂ obtained by a method according to any one of claims 3, 4 6 to 22and 25 and comprising more than 80% TrkCIg₂ monomer.
 55. A preparationof TrkCIg₂ obtained by a method according to any one of claims 3, 4, 6to 22 and 25 and comprising more than 90% TrkCIg₂ monomer.
 56. Apreparation of TrkCIg₂ obtained by a method according to any one ofclaims 3, 4, 6 to 22 and 25 and comprising more than 99% TrkCIg₂monomer.
 57. A preparation of TrkCIg₂ obtained by a method according toany one of claims 3, 4, 6 to 22 and 25 and comprising 100% TrkCIg₂monomer.